Manipulating genomes Flashcards
What is a gene?
A section of DNA that codes for a protein (polypeptide) which results in a characteristic.
What is a locus?
The position on a chromosome where a particular allele is found.
What is a genome?
All of the genetic material an organisms contains.
What were/are the aims of the human genome project?
To work out the order of sequences of all the 3 billion base pairs in the human genome.
To identify all the genes
To develop faster and cheaper methods for sequencing DNA
What is the 1st step in producing a DNA profile?
DNA extraction - DNA is extracted from the tissue sample
What is the 2nd step in producing a DNA profile?
Digestion - Fragment is cut into smaller pieces called satellites by restriction endonucleases. They cut through both strands of DNA. Each type of endonuclease cuts at a specific base sequence called the recognition site. If a mixture of endonuclease are used then predictable satellites will be produced.
Mini satellites are repeats of sequences which are 20-50 base pairs long and micro satellites are 2-4 base pairs long.
What is the 3rd/4th step in producing a DNA profile?
Separation of fragments - fragments need to be separated and shown clearly which is done via electrophoresis. DNA has a slight negative charge and so is able to move when the gel has electricity passed through it. The gel is then immersed in alkali so the strands are broken apart. These single strands are then transferred onto a nylon membrane in excess called southern blotting.
What is the 5th step in producing a DNA profile?
Hybridisation - radioactive or fluorescent DNA probes are added to the membrane. DNA probes are short sequences of DNA that are complimentary to specific sequences of DNA. They bind to DNA under specific conditions of temperature and ph. They attach to particular microsatellites (hybridisation).
What is the 6/7th step in producing a DNA profile?
Development - if radioactive probes were used, an X-ray is taken off the blot. If fluorescent probes are used, then UV light is used to look at the blot. This gives a pattern of bars that can be seen that are specific to the sample taken.
What is PCR and why is it used?
Amplifying DNA - amplifies small amounts of DNA - used to make identical copies of DNA. Can be used on crime scenes - artificial DNA replication. Repeated to give huge numbers. The machine used in this process is called a Thermocycler.
What happens during step 1 of PCR?
Temp is increased to 95 for 30 secs - this breaks the hydrogen bonds between nitrogenous bases breaking apart the strands of DNA.
Samples, polymers, DNA bases and primers are added.
If temp is left high for too long histone proteins around DNA will be damaged.
Starts with 1 strand of DNA.
What happens during step 2 of PCR?
Temp is reduced to 55 - this allows primers to bind (or anneal) to the ends of each DNA strand by complimentary base pairings - choose correct primers.
What happens during step 3 of PCR?
Temp is raised to 72
DNA polymerase attaches at the primer and begins adding complimentary base pairs to the chain - free DNA nucleotides from the original mix. Known as extension - making a complimentary strand of DNA for each strand.
Specialised DNA polymerase called Taq polymerase from thermophilus bacteria is used at these higher temps - our enzymes denature at these temps.
What is needed for PCR?
Excess nucleotides (A,T,C,G)
Primer DNA sequences ( short sequence of DNA that provides a starting point for DNA synthesis
The enzyme DNA polymerase
Sample of DNA
Whats the formula for calculating numbers of DNA strands from PCR?
2^n
n = number of generations or cycles
Why may the calculated value for number of DNA strands from PCR not be achieved in practice?
damage to template
one strand doesn’t separate properly
primers fail to attach
lack of primers/free nucleotides
What is electrophoresis?
Where molecules are separated according to their size/mass and their net overall charge. Different sizes of fragments have different masses, the smaller the fragment the quicker it moves, larger molecules move slower. DNA is negatively charged so its going to move to the anode(+)
What are the steps of electrophoresis?
- DNA is cut into fragments by restriction enzymes
- Pour agarose gel into a gel tray and leave it to solidify
- A row of wells is created at one end of the tray by a comb
- DNA segments loaded into wells with micropipette
- Gel plate is immersed in charged buffer solution - DNA fragments move through gel toward positive electrode - electricity is passed through gel - cathode is near the wells.
- Add probes to make DNA visible - radioactive proves show up under x-ray OR fluorescent show up under UV light.
- The bands of DNA in each sample can be compared for similarities and differences.
What happens during stages 1-2 of DNA sequencing?
DNA template strand chopped up, mixed with primer, bases, free DNA nucleotides, DNA polymerase and terminator bases (STOP) is placed into a thermal cycler. Each type of nucleotide has a fluorescent probe attached. Put temp up and separate DNA into 2 single strands. Turn temp down and allow the primers to anneal to the DNA strand.
What happens during stage 3 of DNA sequencing?
Each time a terminator base is added a strand terminates until all possible chains produced. Largest fragments moves least distance from the +ve electrode,
What happens during stage 4 of DNA sequencing?
Readout from capillary tubes: DNA fragments seperated by electrophasis in capillary tubes by mass and lasers detect the colours and the sequence.
What happens during stage 5 of DNA sequencing?
Computer analysis of all data to give original DNA sequence. Order of colours tells us the order of the bases, - looks like a mass spectrometer.
Whats a benefit of DNA sequencing this way?
Cheaper, quicker and more efficient.
What is the difference between DNA profiling and DNA sequencing?
DNA profiling is the production of genetic fingerprint, unique to a person, based on particular sections of DNA whereas DNA sequencing is a process which determines the precise order bases in DNA sequences.
